The long-term objectives of this project are to focus on the elucidation of of the role of mitochondria in regulating ceramide generation and function in apoptosis in response to many agents that induce ceramide accumulation. Multiple lines of evidence point to roles for ceramide in regulating cell growth in response to extracellular stimuli and stresses and to the participation of ceramide in the response of cells to these stress stimuli. More recent data from the Project Leader and other laboratories and other laboratories are beginning to point to a special role of mitochondrial ceramide and enzymes of ceramide metabolism in the regulation of apoptosis. Based on these results, we propose the following long term hypothesis: Mitochondrial sphingolipid metabolism is an important regulator of the apoptotic response and is intimately related to the action of Bcl-x and Bcl-2. In order to develop this hypothesis, we propose the following specific aims: i) to determine and define ceramide metabolism to mitochondria; ii) to order the pathway from oxidative stress to cytochrome c with respect to ceramide and key members of the Bcl-2 family; and iii) to determine the function of mitochondrial sphingomyelin/ceramide in the activation of the apoptotic program. These studies should allow us to define the existence and regulation of mitochondrial sphingolipid metabolism and its role in the regulation of hepatocyte apoptosis. Defining these novel pathways and mechanisms should provide important insight into livery injury and disease and may provide novel targets for therapeutic intervention.